To date, various methods for producing, purifying, cleaving and using fusion peptides have been explored or reported. These methods are described in some detail below, however, none of this discussion is admitted to represent prior art to the pending claims.
Production
The expression of the synthetic somatostatin gene as a fusion protein in Escherichia coli ("E. coli") is described in Itakura et al., Science, 198:1056-1063 (1977). A number of other recombinant proteins have also been produced as fusion proteins in E. coli, e.g. insulin A and B chain, calcitonin, .beta.-endorphin, urogastrone, .beta.-globin, myoglobin, human growth hormone, and angiotensin. Uhlen and Moks, "Gene Fusions for Purposes of Expression, An Introduction" in Meths. in Enz., 185:129-143 (Academic Press, Inc. 1990).
Shen, "Multiple Joined Genes Prevent Product Degradation in Escherichia Coli" in Proc. Natl. Acad. Sci. USA, 81:4627-4631, 1984 and Shen et al., Canadian Patent No. 1,213,537, describe a method that allows the expression of a stable human proinsulin product in E. coli as encoded by a fused gene construction. Multiple, tandemly linked human proinsulin coding sequences were joined to the 3' side of a fragment containing the lac promoter and the coding sequence for a small part of the NH.sub.2 terminus of .beta.-galactosidase. The polypeptide product of a multiple copy proinsulin gene was then cleaved by a cyanogen bromide treatment into single proinsulin analog moieties having the extra C-terminal pentapeptide Arg-Arg-Asn-Ser-homoserine.
Lennick et al., "High-level expression of .alpha.-human atrial natriuretic peptide from multiple joined genes in Escherichia coli" in Gene, 61:103-112 (1987), describes a method which allows .alpha.-human atrial natriuretic peptide to be synthesized in stable form in E. coli. Eight copies of the synthetic .alpha.-hANP gene were linked in tandem, separated by codons specifying a four amino acid linker with lysine residues flanking the authentic N and C-termini of the 28 amino acid hormone. That sequence was then joined to the 3' end of the fragment containing the lac promoter and the leader sequence coding for the first seven N terminal amino acids of .beta.-galactosidase. The expressed multidomain protein accumulated intracellularly into stable inclusion bodies and was purified by urea extraction of the insoluble cell fraction. The purified protein was cleaved into monomers by digestion with endoproteinase lys-C and trimmed to expose the authentic C-terminus by digestion with carboxypeptidase-B.
Kempe et al., "Multiple-copy genes: production and modification of monomeric peptides from large multimeric fusion proteins", in Gene, 39:239-245 (1985), describes a vector system designed for obtaining polypeptides synthesized in E. coli. Multiple copies of a synthetic gene encoding the neural peptide substance P(SP)(Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH.sub.2) were linked and fused to the lacZ gene. Each copy of the SP gene was flanked by codons for methionine to create sites for cleavage by cyanogen bromide. The isolated multimeric SP fusion protein was converted to monomers of SP analog each containing carboxyl terminal homoserine lactone residue upon treatment with cyanogen bromide in formic acid. The homoserine lactone moiety was subjected to chemical modifications to produce a SP homoserine amide.
Purification
Metal affinity chromatography has recently been used as a basis for protein separations. Arnold, "Metal Affinity Separations: A New Dimension In Protein Processing" in Bio/Technology, 9:151-156 (1991).
Smith et al., "Chelating Peptide-immobilized Metal Ion Affinity Chromatography" in J. Biol. Chem., 263:7211-7215 (1988) describes a specific metal chelating peptide on the NH.sub.2 terminus of a protein that can be used to purify that protein using immobilized metal ion affinity chromatography. Recombinant fusions consisting of trpLE'-proinsulin or LHRH analogs were cleaved with cyanogen bromide thus exposing a N-terminal His-Trp Ni-affinity tag which was used to purify the smaller peptide hormones in 7M urea.
Hochuli et al., "Genetic Approach to Facilitate Purification of Recombinant Proteins With A Novel Metal Chelate Adsorbent", in Bio/Technology, 11:1321-1325 (1988), describe a polyhistidine peptide containing 2-6 adjacent histidines fused to mouse dihydropholate reductase at both the C and N-termini. The fusion proteins were purified on Ni(II)-NTA and subsequently treated with carboxypeptidase A to remove the polyhistidine tail. Fusion protein with the 6 histidine tail was extracted from E. coli with 6M GuHCl and loaded without further purification on a Ni(II)-NTA column.
Cleavage
Several chemical and enzymatic agents have been used to specifically cleave fusion proteins including cyanogen bromide, formic acid, hydroxylamine, collagenase, enterokinase factor X.sub.A, thrombin, trypsin, clostripain and ala-subtilisin. Uhlen and Moks, Meths. in Enz., 185:129-143 (1990) and Emtage, "Biotechnology & Protein Production" in Delivery Systems for Peptide Drugs, pp. 23-33 (1986).
Use
Kempe et al., "Multiple-copy genes: production and modification of monomeric peptides from large multimeric fusion proteins", in Gene, 39:239-245 (1985), describes the production of a SP peptide homoserine amide. A SP analog containing a carboxyl terminal homoserine lactone residue was treated with 30% NH.sub.4 OH at room temperature for 30 minutes and also with methanolic NH.sub.3 (10%).
Horn et al., FEBS Letters, 36:285-288 (1973) and Horn, Analytical Biochemistry, 69:583-589 (1975) describe a procedure for attaching cyanogen bromide peptides to resins by their C-terminal homoserine residues. The method involves lactonization of the homoserine residue with trifluoroacetic acid and subsequent aminolysis of the lactone with an amino resin.
Calloway et al., Antimicrobial Agents and Chemotherapy, 37:1614-1619 (1993) describes the production of cecropin A with a C-terminal homoserine that was chemically modified to produce a recombinant peptide with similar activity to that of cecropin A produced by cecropia pupae.